Chromogenic compositions comprise indicative compounds capable of producing colour by causing a displacement of, or the appearance of, absorbent bands in the visible spectrum. Chromogenic compositions have significant applications in the fields of histology, molecular biology, microbiology and the like.
Histological examination of biological samples, such as tissue samples, is commonly performed using manual or automated processes. Specialized equipment and protocols have been developed for many automated stains. There remains a need for automation of non-routine, complex or special stains. Furthermore, the increase in the use of multiple stains on a single tissue sample has required an increase in the range of stains that are distinguishable from other stains, and which can be successfully incorporated into an automated protocol.
The development of stains for both manual and automated use is often limited because of the instability of the chromogenic composition or components of the chromogenic composition, the cost of existing solutions, shelf life and storage requirements such as the need for refrigeration of some reagents.
Diazonium salts have been suggested for use in preparing chromogenic compositions. Some diazonium salts are hazardous and require careful handling when dry.
Diazonium salts are normally hygroscopic and therefore will absorb moisture from the air and become unstable if not kept in tightly sealed containers. Diazotized aromatic amines used as colorimetric indicators are also unstable in solution. While stability of these solutions can be enhanced by storage of the solution in cold and dark conditions, even under these conditions significant deterioration as evidenced by discoloration, precipitate formation, and loss of reactivity can be seen in a relatively short time.
Due to the disadvantages of using diazonium salt compounds directly, diazonium-based reagents are prepared by combining precursor reagents. However, these diazonium-based reagents are unstable and must be used within a few days or discarded.
Approaches to improve the stability of chromogenic compositions, such as diazonium-based reagents, have been modest. Accordingly, it would be very desirable to develop diazonium chromogenic compositions having even more pronounced stability.
Any discussion of documents, devices, acts or knowledge in this specification is included to explain the context of the invention. It should not be taken as an admission that any of the material formed part of the prior art base or the common general knowledge in the relevant art on or before the priority date of the invention disclosed herein or, any claims defined herein.